ABCMdb

ABCC8 p.Glu1506Asp

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (91%), F: D (91%), G: D (95%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (91%), P: D (85%), Q: D (85%), R: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Mutations of the same conserved glutamate residue ... Diabetes. 2011 Jun;60(6):1813-22. Mannikko R, Flanagan SE, Sim X, Segal D, Hussain K, Ellard S, Hattersley AT, Ashcroft FM
Mutations of the same conserved glutamate residue in NBD2 of the sulfonylurea receptor 1 subunit of the KATP channel can result in either hyperinsulinism or neonatal diabetes.
Diabetes. 2011 Jun;60(6):1813-22., [PMID:21617188]

Abstract [show]
OBJECTIVE: Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K(+) channel (K(ATP) channel) sulfonylurea receptor 1 (SUR1) subunit were detected heterozygously in patients with neonatal diabetes. A mutation at the same residue (E1506K) was previously shown to cause congenital hyperinsulinemia. We sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia. RESEARCH DESIGN AND METHODS: Neonatal diabetic patients were sequenced for mutations in ABCC8 (SUR1) and KCNJ11 (Kir6.2). Wild-type and mutant K(ATP) channels were expressed in Xenopus laevis oocytes and studied with electrophysiological methods. RESULTS: Oocytes expressing neonatal diabetes mutant channels had larger resting whole-cell K(ATP) currents than wild-type, consistent with the patients' diabetes. Conversely, no E1506K currents were recorded at rest or after metabolic inhibition, as expected for a mutation causing hyperinsulinemia. K(ATP) channels are activated by Mg-nucleotides (via SUR1) and blocked by ATP (via Kir6.2). All mutations decreased channel activation by MgADP but had little effect on MgATP activation, as assessed using an ATP-insensitive Kir6.2 subunit. Importantly, using wild-type Kir6.2, a 30-s preconditioning exposure to physiological MgATP concentrations (>300 micromol/L) caused a marked reduction in the ATP sensitivity of neonatal diabetic channels, a small decrease in that of wild-type channels, and no change for E1506K channels. This difference in MgATP inhibition may explain the difference in resting whole-cell currents found for the neonatal diabetes and hyperinsulinemia mutations. CONCLUSIONS: Mutations in the same residue can cause either hyperinsulinemia or neonatal diabetes. Differentially altered nucleotide regulation by NBD2 of SUR1 can explain the respective clinical phenotypes.

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No. Sentence Comment
0 Mutations of the Same Conserved Glutamate Residue in NBD2 of the Sulfonylurea Receptor 1 Subunit of the KATP Channel Can Result in Either Hyperinsulinism or Neonatal Diabetes Roope Männikkö,1 Sarah E. Flanagan,2 Xiuli Sim,1 David Segal,3 Khalid Hussain,4 Sian Ellard,2 Andrew T. Hattersley,2 and Frances M. Ashcroft1 OBJECTIVE-Two novel mutations (E1506D, E1506G) in the nucleotide-binding domain 2 (NBD2) of the ATP-sensitive K+ channel (KATP channel) sulfonylurea receptor 1 (SUR1) subunit were detected heterozygously in patients with neonatal diabetes. Login to comment
50 Furthermore, the mutation of E1506 to lysine (E1506K) results in reduced channel activation by MgADP and is associated with hyperinsulinism (26,27). Login to comment
51 This article reports our investigation of how the mutation of E1506 to aspartate (E1506D) or glycine (E1506G) results in the opposite clinical condition of neonatal diabetes. Login to comment
96 A small but significant increase in resting current was observed for both homomeric Kir6.2/SUR1-E1506D (homE1506D) and Kir6.2/SUR1-E1506G (homE1506G) channels (Fig. 1). Login to comment
100 The resting currents of heterozygous (het) E1506D and hetE1506G channels were significantly greater than wild-type channels, which may explain why these mutations cause neonatal diabetes. Login to comment
111 This suggests that the neonatal diabetes mutations have little (E1506D) or no (E1506G) effect on expression levels of the KATP channel in the heterozygous state. Login to comment
115 A-C: Representative whole-cell current amplitudes evoked by repeated voltage steps from 210 to 230 mV for wild-type (A, WT), and homomeric (B, homE1506D) or heterozygous (C, hetE1506D) Kir6.2/SUR1-E1506D channels. Login to comment
136 B: Kir6.2/SUR1-E1506D (n = 11), IC50 = 20.1, h = 1.05. Login to comment
138 D: Kir6.2/SUR1-E1506K (n = 11), IC50 = 11.0, h = 1.14. Login to comment
139 E: Kir6.2/SUR1-E1506D (n = 7), IC50 = 8.2, h = 1.00. Login to comment
179 No increase in current was observed for the Kir6.2-G334D/SUR1-E1506D (G334D/E1506D) or Kir6.2-G334D/SUR1-E1506G (G334D/ E1506G) channels; in contrast, the Kir6.2-G334D/SUR1-E1506K (G334D/E1506K) currents increased 1.5-fold on excision. Login to comment
180 This suggests that the G334D/E1506 and G334D/ E1506K channels were partially blocked under resting conditions in the oocyte, whereas the G334D/E1506D and G334D/E1506G channels were fully open. Login to comment
181 Larger amplitudes were found for the G334D/E1506 (8.5 6 1.9 nA, n = 16) and G334D/E1506K (14 6 1.9 nA, n = 9) currents than for the G334D/E1506D (1.0 6 0.2 nA, n = 11) or G334D/ E1506G (0.8 6 0.2 nA, n = 13) currents, again suggesting that the E1506D and E1506G mutations may impair surface expression in the homomeric state. Login to comment
193 The error bars show the SEM. E: Representative current traces for WT and Kir6.2/SUR1-E1506D (E1506D) channels. Login to comment
195 The WT trace is interrupted to align the time point of ATP removal with that of the E1506D trace. Login to comment
204 The off-rate of MgADP was not significantly different for the G334D/E1506K channels but was slower for the G334D/E1506D and G334D/E1506G channels, with a toff of 10 and 11 s, respectively. Login to comment
205 The off-rate of MgATP was significantly less for all three mutant channels, with a toff of 8.5 s for G334D/E1506K, 16 s for G334D/E1506G, and 67 s for G334D/E1506D. Login to comment
210 Such ATP preconditioning reduced the ATP sensitivity of the two neonatal diabetic mutant channels but had little or no effect on wild-type or E1506K channels, respectively (Table 1). Login to comment
211 As a consequence, the IC50 for the ATP block of the homomeric and heterozygous E1506D and E1506G channels was significantly greater than wild-type, whereas that of hetE1506K was no different, and that of homE1506K was actually slightly smaller (Fig. 6, Table 1). Login to comment
213 Preconditioning pulses of .300 mmol/L MgATP markedly reduced the ability of 100 mmol/L MgATP to block E1506D and E1506G channels but had only a small effect on wild-type channels and no effect on the homE1506K channels. Login to comment
252 Mutation of the equivalent residue in MRP1 to aspartate does not alter MgADP binding (32), which suggests that SUR1-E1506D might bind MgADP but that binding no longer leads to channel activation. Login to comment
264 The slower off-rate of MgATP seen with the neonatal diabetes mutations (especially E1506D) may indicate that the channel becomes trapped in a particular state of the reaction cycle that is associated with increased channel activity; for example, the MgATP-bound or MgADP + Pi state. Login to comment
265 The off-rate of MgADP was much faster than that of MgATP for the E1506D channels, and MgADP had little stimulatory effect, which supports arguments that it cannot be the MgADP-bound state. Login to comment
266 The most striking difference between the E1506K and E1506G/E1506D channels is that shown in Figs. 6 and 7: pre-exposure to millimolar concentrations of MgATP desensitizes the channel to subsequent inhibition by a lower ATP concentration. Login to comment
95 A small but significant increase in resting current was observed for both homomeric Kir6.2/SUR1-E1506D (homE1506D) and Kir6.2/SUR1-E1506G (homE1506G) channels (Fig. 1). Login to comment
99 The resting currents of heterozygous (het) E1506D and hetE1506G channels were significantly greater than wild-type channels, which may explain why these mutations cause neonatal diabetes. Login to comment
110 This suggests that the neonatal diabetes mutations have little (E1506D) or no (E1506G) effect on expression levels of the KATP channel in the heterozygous state. Login to comment
114 A-C: Representative whole-cell current amplitudes evoked by repeated voltage steps from 210 to 230 mV for wild-type (A, WT), and homomeric (B, homE1506D) or heterozygous (C, hetE1506D) Kir6.2/SUR1-E1506D channels. Login to comment
135 B: Kir6.2/SUR1-E1506D (n = 11), IC50 = 20.1, h = 1.05. Login to comment
178 No increase in current was observed for the Kir6.2-G334D/SUR1-E1506D (G334D/E1506D) or Kir6.2-G334D/SUR1-E1506G (G334D/ E1506G) channels; in contrast, the Kir6.2-G334D/SUR1-E1506K (G334D/E1506K) currents increased 1.5-fold on excision. Login to comment
192 The error bars show the SEM. E: Representative current traces for WT and Kir6.2/SUR1-E1506D (E1506D) channels. Login to comment
194 The WT trace is interrupted to align the time point of ATP removal with that of the E1506D trace. Login to comment
203 The off-rate of MgADP was not significantly different for the G334D/E1506K channels but was slower for the G334D/E1506D and G334D/E1506G channels, with a toff of 10 and 11 s, respectively. Login to comment
212 Preconditioning pulses of .300 mmol/L MgATP markedly reduced the ability of 100 mmol/L MgATP to block E1506D and E1506G channels but had only a small effect on wild-type channels and no effect on the homE1506K channels. Login to comment
251 Mutation of the equivalent residue in MRP1 to aspartate does not alter MgADP binding (32), which suggests that SUR1-E1506D might bind MgADP but that binding no longer leads to channel activation. Login to comment
263 The slower off-rate of MgATP seen with the neonatal diabetes mutations (especially E1506D) may indicate that the channel becomes trapped in a particular state of the reaction cycle that is associated with increased channel activity; for example, the MgATP-bound or MgADP + Pi state. Login to comment

[hide] Neonatal Diabetes and Congenital Hyperinsulinism C... Front Endocrinol (Lausanne). 2015 Apr 15;6:48. doi: 10.3389/fendo.2015.00048. eCollection 2015. Ortiz D, Bryan J
Neonatal Diabetes and Congenital Hyperinsulinism Caused by Mutations in ABCC8/SUR1 are Associated with Altered and Opposite Affinities for ATP and ADP.
Front Endocrinol (Lausanne). 2015 Apr 15;6:48. doi: 10.3389/fendo.2015.00048. eCollection 2015., [PMID:25926814]

Abstract [show]
ATP-sensitive K(+) (KATP) channels composed of potassium inward-rectifier type 6.2 and sulfonylurea receptor type 1 subunits (Kir6.2/SUR1)4 are expressed in various cells in the brain and endocrine pancreas where they couple metabolic status to membrane potential. In beta-cells, increases in cytosolic [ATP/ADP]c inhibit KATP channel activity, leading to membrane depolarization and exocytosis of insulin granules. Mutations in ABCC8 (SUR1) or KCNJ11 (Kir6.2) can result in gain or loss of channel activity and cause neonatal diabetes (ND) or congenital hyperinsulinism (CHI), respectively. SUR1 is reported to be a Mg(2+)-dependent ATPase. A prevailing model posits that ATP hydrolysis at SUR1 is required to stimulate openings of the pore. However, recent work shows nucleotide binding, without hydrolysis, is sufficient to switch SUR1 to stimulatory conformations. The actions of nucleotides, ATP and ADP, on ND (SUR1E1506D) and CHI (SUR1E1506K) mutants, without Kir6.2, were compared to assess both models. Both substitutions significantly impair hydrolysis in SUR1 homologs. SUR1E1506D has greater affinity for MgATP than wildtype; SUR1E1506K has reduced affinity. Without Mg(2+), SUR1E1506K has a greater affinity for ATP(4-) consistent with electrostatic attraction between ATP(4-), unshielded by Mg(2+), and the basic lysine. Further analysis of ND and CHI ABCC8 mutants in the second transmembrane and nucleotide-binding domains (TMD2 and NBD2) found a relation between their affinities for ATP (+/-Mg(2+)) and their clinical phenotype. Increased affinity for ATP is associated with ND; decreased affinity with CHI. In contrast, MgADP showed a weaker relationship. Diazoxide, known to reduce insulin release in some CHI cases, potentiates switching of CHI mutants from non-stimulatory to stimulatory states consistent with diazoxide stabilizing a nucleotide-bound conformation. The results emphasize the greater importance of nucleotide binding vs. hydrolysis in the regulation of KATP channels in vivo.

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Sentences [show]
No. Sentence Comment
37 The E1506D substitution increases the affinity of SUR1 for MgATP while E1506K reduces affinity. Login to comment
39 Both substitutions reduce affinity for MgADP, consistent with electrophysiological data indicating that E1506D and E1506K produce channels that are less sensitive to stimulation by MgADP. Login to comment
91 Two substitutions, E1506D and E1506K, causes of ND and CHI, respectively, have opposite effects on the affinity for MgATP Several ND mutations in SUR1 increase the apparent affinity for ATP (8, 9). Login to comment
92 To extend these observations two SUR1 substitutions, E1506D and E1506K, well studied at the electrophysiological level (15) and identified with ND and CHI, respectively, were analyzed. Login to comment
100 Wildtype SUR1 is potentially in a steady-state, slowly hydrolyzing MgATP, while the E1506D and E1506K substitutions are both expected to impair hydrolysis. Login to comment
102 Comparison of the approximate EC50 values, 50 mM, 900 &#b5;M and 10 &#b5;M for E1506K, WT and E1506D, respectively, suggests there is an ~5000-fold range in affinities. Login to comment
119 10-2 10-1 100 101 102 103 104 105 0.0 0.2 0.4 0.6 0.8 1.0 WT E1506D E1506K E1506Q Specific Bound GBC [MgATP] (&#b5;M) 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0.0 0.2 0.4 0.6 0.8 1.0 WT E1506K E1506D E1506Q Specific Bound GBC [ATP 4- ] (&#b5;M) B A C FIGURE 2 | (A) Representation of NBD2 based on Sav1866. Login to comment
122 to the current regulatory model, both E1506 substitutions have reduced affinity for MgADP (Figure 4), consistent with electrophysiological data demonstrating that SUR1E1506D/Kir6.2 and 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0.0 0.2 0.4 0.6 0.8 1.0 E1506Q Q1178R E1506D R1182Q I1424V WT S1185A C1174F E1506K G1479R Specific Bound GBC [MgATP] (&#b5;M) 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0.0 0.2 0.4 0.6 0.8 1.0 E1506Q E1506K Q1178R I1424V E1506D R1182Q WT S1185A C1174F G1479R Specific Bound GBC [ATP 4- ] (&#b5;M) B A FIGURE 3 | Comparison of nucleotide-induced conformational switching in WT and SUR1 mutants. Login to comment
151 Figure 5 shows that diazoxide potentiates the 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 Q1178R I1424V R1182Q S1185A C1174F WT E1506Q E1506D G1479R E1506K Specific Bound GBC [MgADP] (&#b5;M) FIGURE 4 | MgADP-induced conformational switching in WT and SUR1 mutants. Login to comment
170 It is notable that the E1506D, and particularly the E1506Q, substitutions with higher affinities for ATP are not switched appropriately by endogenously generated ATP. Login to comment